Use of peptides

ABSTRACT

We claim a therapeutic method of inducing programmed cell death comprising administering to a recipient a peptide of 10–25 amino acids, comprising the sequence: (KR)xxYxxx(F/Q)L(L/M) wherein x is any amino acid.

RELATED APPLICATION INFORMATION

This application claims priority under 35 U.S.C. § 371 from PCTApplication No. PCT/GB00/02414 (published under PCT Article 21(2) inEnglish), filed on Jun. 21, 2000, which claims the benefit of GreatBritain Application Serial No. 9914480.0, filed on Jun. 21, 1999, thedisclosures of which are incorporated by reference herein in theirentireties.

The present invention relates to eukaryotic Initiation Factor 4G(eIF4GI, GII) and derivatives of eIF4E Binding Proteins (4-E-BP1, 2, 3,4) that interact with it.

By way of introduction, the proposed mechanism of eukaryotic initiationfactor complex formation will be described with reference to FIG. 1. TheeIF4F complex is capable of initiating translation of 5′ capped (m⁷G)mRNAs¹. This complex comprises eIF3, eIF4A, eIF4E and eIF4G (FIG. 1).

-   -   eIF4G acts as a scaffold around which the other components are        assembled.    -   eIF4A is a helicase which is required to unwind regions of        secondary structure in the 5′UTR of the mRNA.    -   eIF3 is responsible for recruiting the 40S ribosomal sub-unit to        the complex, interacting with both the 40S ribosomal sub-unit        and eIF4G.

eIF4E binds to both eIF4G and to the m⁷G cap at the 5′ end of the mRNA,hence recruiting the 40S ribosomal sub-unit to the 5′ untranslatedregion (UTR) of capped mRNAs.

eIF4E independent routes exist for the initiation of translation of somemessages² (eg. via an internal ribosome entry site (IRES)). However,mRNAs containing a long 5′ UTR are dependent on eIF4E for therecruitment of the eIF4F complex to the m⁷G cap, and the subsequentunwinding of the UTR by eIF4A. The critical role of eIF4E in capdependent translation is attributed to the limited availability of theactive species. eIF4E appears to be in limiting amounts relative toother eIF4F components¹, requires phosphorylation (by Mnk 1³) formaximum activity and can be excluded from the eIF4F complex by bindingto a 4E-BP^(4,5) (FIG. 2).

There is increasing evidence for a role of eIF4E in carcinogenesis.eIF4E induces cap dependent translation initiation in response to anumber of mitogenic or proliferative stimuli^(1,4,6). Hormone and growthfactor induced signal transduction can lead to hyperphosphorylation of4E-BP by mTOR, resulting in the release of 4E-BP-bound eIF4E mTOR,resulting in the release of 4E-BP-bound eIF4E (FIG. 2). Similar stimulialso lead to activation of eIF4E via phosphorylation by Mnk-1. Theresultant increase in eIF4E activity is required for the translation ofseveral cap-dependent transcripts whose translation products arerequired for proliferation (eg. cyclin D1⁷, Ornithine Decarboxylase(ODC)³).

The number of reports of increased levels of eIF4E in tumour samples isgrowing steadily^(9,10), and in some cases eIF4E levels have beenproposed to be a good indicator of prognosis^(11,12). Overexpression ofeIF4E in cultured cell lines is reported to result in a transformedphenotype^(13,14.)

Overall these results have suggested that inhibiting eIF4E would resultin inhibition of cap-dependent translation, resulting in little or noexpression of mRNAs with strong eIF4E dependency for translation. Thisis expected to cause reduction in expression of several proteinsinvolved in proliferation, and to reduce the transformed phenotype ofsome tumour cells.

It has also been reported that overexpression of eIF4E is capable ofacting as an anti-apoptotic survival signal in fibroblasts undergoingMyc-induced apoptosis in serum-restricted conditions¹⁵.

The variety of eIF4E interacting proteins (eIF4G and 4E-BPs) has allowedidentification of a common motif, (K/R)xxYDRxFL(L/M), required forbinding to eIF4E⁴. Subsequently a 20 amino acid fragment of human 4E-BP1containing this motif was shown to be capable of binding to recombinantmouse eIF4E and inhibiting cap-dependent translation in an in vitrotranslation assay¹⁶, presumably by disrupting the formation of the eIF4Fcomplex.

The proposed approach was to use eIF4E-binding peptides (derived fromeIF4G and 4E-BPs) to inhibit formation of the eIF4F complex and reducecap-dependent translation (FIG. 3).

The present invention is based upon the observation that eIF4E bindingpeptides have been shown for the first time to induce programmed celldeath. This observation is surprising given that the expected effect ofsuch peptides was to reduce expression of several proteins involved inproliferation, resulting in growth inhibition of, or increasedcytotoxicity to tumour cells. This surprising observation renders thesepeptides of utility in therapy.

Thus, in a first aspect the present invention provides the use of eIF4Ebinding agents, such as peptides or peptidemimetics in therapy, moreparticularly for the induction of programmed cell death. Particularpeptides found to be capable of inducing programmed cell death include asequence of human eIF4G₅₆₉₋₅₈₀, wheat eIF4G₆₂₋₇₃ and humaneIF4E-BP(1&2)₅₁₋₆₂ and derivatives and fragments thereof. Numberingaccording to Accession numbers AF104913, M95746, NM_(—)004095 andNM_(—)004096 respectively.

Thus the peptides of use in the present invention include the sequences;

human eIF4G₅₆₉₋₅₈₀, KKRYDREFLLGF [SEQ ID NO: 1]

wheat eIF4G₆₂₋₇₃ RVRYSRDQLLDL [SEQ ID NO: 2] and,

human eIF4E-BP(1&2)₅₁₋₆₀ RIIYDRKFL(L/M) [SEQ ID NO: 3], and variants orderivatives thereof. A consensus may be derived from the above threesequences.

Thus, in a further aspect the present invention provides use of apeptide comprising a sequence:

YxxxxLØ [SEQ ID NO: 4]

wherein x is a variable amino acid and Ø is Leu, Met or Phe;

or a fragment or derivative thereof in therapy, more particularly forthe induction of programmed cell death.

Alternatively the peptide may comprise the sequence:(K/R)xxYxxx(F/Q)L(L/M) [SEQ ID NO: 5]

It is to be understood that “K/R” refers to an amino acid which iseither lysine (K) or arginine (R), “x” may be any of the 20 amino acidsor may be a synthetic or unnatural amino acid, “F/Q” refers to an aminoacid which is either phenylalamine (F) or glutamine (Q) and “L/M” refersto an amino acid which is either leucine (L) or methionine (M). Theremainder of the sequence is understood to relate to the standard singleletter symbol for amino acids.

Particular sequences may include

KKRYDREFLLGF [SEQ ID NO: 1] (human eIF4G₄₁₃₋₄₂₄),

RVRYSRDQLLDL [SEQ ID NO: 2] (wheat eIF4G₆₂₋₇₃) and

RIIYDRKFL(L/M) [SEQ ID NO: 3] (human eIF4E-BP₅₁₋₆₀).

The invention also relates to the use of fragments and derivatives ofthese peptides. Fragments are defined herein as any portion of thepeptides described that substantially retain the activity of the parentpeptide. Derivatives are defined as any modified forms of said peptideswhich also substantially retain the activity of the parent peptide. Suchderivatives may take the form of amino acid substitutions which may bein the form of like for like eg. a polar amino acid residue for anotherpolar residue or like for non-like eg. substitution of a polar aminoacid residue for a non-polar residue as discussed in more detail below.

Thus, the present invention further provides derivatives of thesequences disclosed above for use in the induction of cell death.

Replacement amino acid residues may be selected from the residues ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. The replacement amino acid residue may additionally be selectedfrom unnatural amino acids. Within the above definitions of the peptidecarrier moieties of the present invention, the specific amino acidresidues of the peptide may be modified in such a manner that retainstheir ability to induce programmed cell death, such modified peptidesare referred to as “variants”. Thus, homologous substitution may occuri.e. like-for-like substitution such as basic for basic, acidic foracidic, polar for polar, etc. Non-homologous substitution may also occurie. from one class of residue to another or alternatively involving theinclusion of unnatural amino acids such as ornithine (O), diaminobutyricacid (B), norleucine (N), pyriylalanine, thienylallanine,naphthylalanine and phenylglycine and the like. Within each peptidecarrier moiety more than one amine acid residue may be modified at atime, but preferably when the replacing amino acid residue is alanine,less than 3.

As used herein, amino acids are classified according to the followingclasses;

basic; H,K,R

acidic; D,E

polar; A,F,G,I,L,M,P,V,W

non-polar; C,N,Q,S,T,Y,

(using the internationally accepted amino acid single letter codes)

and homologous and non-homologous substitution is defined using theseclasses. Thus, homologous substitution is used to refer to substitutionfrom within the same class, whereas non-homologous substitution refersto substitution from a different class or by an unnatural amino acid.

In general, the term “peptide” refers to a molecular chain of aminoacids with the defined biological activity. If required, it may bemodified in vivo and/or in vitro, for example, by glycosylation,myristoylation, amidation, carboxybolation or phosphorylation. Thusinter alia peptides, oligopeptides and polypeptides are included. Thepeptides disclosed herein may be obtained, for example, by synthetic orrecombinant techniques known in the art.

The term also extends to cover, for example, polypeptides which containany of the above disclosed sequences and, in particular, whereinbiological activity, that is, the polypeptide is capable of binding toeIF4E protein, is retained. Typically the length of the peptides of thepresent invention are between 7–25 amino acids in length, morepreferably 10–20 amino acids in length.

In a further aspect the present invention provides use of a peptidecomprising sequence:

YxxxxLØ [SEQ ID NO: 4] wherein x is a variable amino acid and Ø is Leu,Met or Phe;

or fragment or derivate thereof in the manufacture of a medicament fortherapy, more particularly for inducing cell death.

In particular, the peptide is used to induce the cell death in tumourcells.

In yet a further aspect, the present invention provides a polynucleotidefragment encoding a peptide comprising sequence:

YxxxxLØ [SEQ ID NO: 4] wherein x is a variable amino acid and Ø is Leu,Met or Phe.

“Polynucleotide fragment” as used herein refers to polymeric form ofnucleotides of any length, both to ribonucleic acid sequence and todeoxyribonucleic acid sequences. In principal, this term refers to theprimary structure of the molecule, thus this term includes doublestranded and single stranded DNA, as well as double and single strandedRNA, and modifications thereof.

As described above, the presence of a peptide comprising the abovesequences can induce programmed cell death (apoptosis) in mammaliancells. The peptides of the present invention therefore have utility intreating diseases associated with undesirable cellproliferation/neoplasia. In particular the peptides have utility asanticancer or antitumour agents. Therefore, it may be desirable todirect the peptides to the site of action ie. the tumour. Thus, in thecase of peptides, they may be conjugated to or associated with celland/or tumour targeting agents, or in the case of the polynucleotidefragments provided as an expression cassette which comprises apolynucleotide sequence which encodes any of the above disclosedpeptides, and a tumour-specific inducible promoter which would allowexpression of the peptide of the present invention only in tumour cells.The peptides of the present invention may also be conjugated orassociated with agents designed to facilitate uptake into cell such astransport peptides eg. penetratin.

The present invention also relates to the use of peptidemimetics whichbind eIF4E and function to induce programmed cell death. Suchpeptidemimetics are generally small molecules which function in the samemanner as the peptides disclosed herein.

These and other aspects of the present invention will become apparentfrom the following description when taken in combination with theaccompanying Figures, in which:

FIGS. 1 to 3 are diagrams illustrating the interaction of eIF4G andeIF4E;

FIG. 4 illustrates the binding of human eIF4G-Penetratin conjugate toeIF4E and the sequences employed;

FIG. 5 illustrates the binding of human, yeast, wheat and scrambledeIF4G, human 4E-BP1 and 4E-BP2 to eIF4E and the sequences employed;

FIG. 6 illustrates human 4E-BP1 competing with eIF4G peptides for thebinding of eIF4E and the sequences employed;

FIG. 7 illustrates wheat eIF4G₍₆₂₋₇₃₎ inhibiting cap-dependenttranslation initiation;

FIG. 8 is a table illustrating the induction of apoptosis by eIF4Gpeptides in MRC5 cells;

FIG. 9 is a summary of the results of inhibition ofeIF4G₍₄₁₃₋₄₂₄₎-induced apoptosis;

FIG. 10 is a summary of the results of inhibition ofeIF4G₍₅₆₉₋₅₈₀₎-induced apoptosis in MRC5 cells overexpressingconstitutively active MEK/ERK;

FIG. 11 is a diagram illustrating the interaction of eIF4G and eIF4E;

FIG. 12 illustrates that eIF4E-binding peptides leads to rapid,dose-dependent, cell death. a). Sequences of biotinylated,penetratin-linked peptides. U=Norvaline, a substitution for cysteine.Conserved residues that are important in binding to eIF4E areunderlined. b). In vitro binding assay. i) 1 μg recombinant eIF4E wasincubated with 0.2 mM biotinylated peptides in a total volume of 50 μlwash buffer (1×PBS/250 mM Kcl) for 1 h at 4° C. ii) 0.2 mM Hu4G or Hu4GYLL-AAA was incubated with 200 μg MRC5 cell lysate for 1 h at 4° C. in atotal volume of 50 μl wash buffer. In all cases the biotinylatedpeptides and associated proteins were pulled down using streptavidinagarose. The proteins were separated by SDS PAGE and subjected toWestern blotting using anti-eIF4E antibody. Detection was by ECL;

FIGS. 13 a–d are graphs showing cell survival (% of control, untreatedcells) as measured by MTT assay. a) Lane 1: 10 μM BP2 peptide added toserum-fed cells or Lane 2, 3, 4: 10 μM BP2 peptide added to 24 h, 48 hor 72 h serum-starved cells respectively. Lane 5: 10 μM BP2 YLL-AAAadded to 72 h serum starved cells. Lane 6: 72 h serum starved cellsincubated in 10% serum for 1 h prior to addition to 10 μM BP2 peptide.Lane 7: 72 h serum starved cells incubated with 100 μg/ml cycloheximidefollowed by a 1 h incubation with 10% serum prior to the addition of 10μM BP2 peptide. In all cases the cells were then further incubated for 1h in 0.1 mg/ml MTT. Cells were lysed in DMSO and absorbance was measuredat 570 mm. b–d) Varying concentrations of peptides were added to 72 hserum starved MRC5 cells. After 30 min incubation the cells were thenfurther incubated for 1 h in 0.1 mg/ml MTT. Cells were lysed in DMSO andabsorbance was measured at 570 nm. All these results are representativeof three separate experiments;

FIG. 14 illustrates that cell death induced by eIF4E-binding peptidesshows characteristics of apoptosis. a) Time lapse images ofserum-starved MRC5 cells treated with 10 μM Hu4G peptide for theindicated times. b) FACS analysis of MRC5 cells treated with 20 μM Hu4Gor Hu4G YLL-AAA for 40 min. c) TUNEL analysis using the “In situ celldeath detection kit” (Boehringer Mannheim). MRC5 cells were incubatedwith 10 μM Hu4G peptide for 10 min. Cells visualised with fluorescein byfluorescence microscopy. No signal was observed in untreated cells orcells treated with 10 μM Hu4G YLL-AAA peptide (results not shown). 80%of cells incubated with the 10 μM Hu4G peptide fluoresced positive. d)Images of DAPI-stained MRC5 cells incubated with either 20 μM Hu4G orHu4G YLL-AAA peptide for 40 min. Arrows indicate the position of a cellwith either a condensed nucleus (top right) or a nucleus with a punctateappearance (bottom left). e) Visualisation of effects on the MPT.Serum-starved MRC5 cells were loaded with 0.1 μM JCl for 30 min prior toaddition of 10 μM peptide. Changes in mitochondrial permeability wereviewed by laser scanning confocal microscopy (krypton/argon laser) usingidentical setting. Green channel: excitation 488 nm/emission 522 nm. Redchannel: excitation 568 nm/emission 585 nm. Channels were collectedseparately to avoid cross over. Images were taken 5 min after additionof the peptide;

FIG. 15 illustrates that the acute activation of MAP kinase protectscells from 4E-binding peptide induced cell death; and

FIG. 16 illustrates that eIF4E binding peptide cell death is not througheIF4E's known role in mRNA translation. a) 72 h serum-starved MRC5 cellswere treated with 100 μg/ml and 10 μg/ml cylcoheximide, 1 μg/ml and 0.1μg/ml pactamycine or 10 μM Hu4G peptide for 2 h prior to a MTT assay.Cells were pulsed labelled with [³⁵S]methionine for 30 min afteraddition of the inhibitors/peptide. The incorporation of [³⁵S]methionineinto protein was determined following hot trichloroacetic acidprecipitation. b) 72 h serum-starved MRC5 cells were treated with 100μg/ml cycloheximide (Chx), 1 μg/ml pactamycine (Pact) or c) MRC5 cellswhich had been starved of serum for 72 h were pre-incubated with 100μg/ml cycloheximide or 1 μg/ml pactamycine for 30 min prior to additionof 10 μM Hu4G peptide. Cells were then further incubated for 1.5 h priorto a MTT assay. Results from three independent experiments (+/− SEM).

EXAMPLES

Abbreviations.

Amino acid and peptide nomenclature conforms to IUPAC-IUB rules (Eur. J.Biochem. 1984, 138, 9–37). Other abbreviations: Ahx, 6-aminohexanoyl;APase, alkaline phosphatase; DE MALDI-TOF MS, delayed-extractionmatrix-assisted laser desorption ionisation time-of-flight massspectrometry; DIEA, N,Ndiisopropylethylamine; PBS, phosphate-bufferedsaline (10 mM phosphate, 150 Mm NaCl, pH 7.4); PyBOP,Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate;RP-HPLC, reversed-phase high-performance liquid chromatography; TFA,trifluoroacetic acid.

Material and Methods

General

The peptide deprotection/cleavage mixture used throughout was asfollows: 0.75:0.5:0.5:0.25:10 (W/V/V/V/V) PhOH, H₂O, PhSMe,1,2-ethanedithiol, TFA (Beavis, R. C., et al., (1992) Organic MassSpectrometry 27, 156–158). Analytical and preparative RP-HPLC wasperformed using Vydac 218TP54 (4.6×250 mm) and 218TP1022 (22×250 mm)columns, respectively. Flow rates of 1 mL/min for analytical runs and 9mL/min for preparative work were used (at 25° C.). Gradient elution withincreasing amounts of MeCN in H₂O (containing 0.1% TFA) over 20 min(anal.) and 40 min (prep.) was performed. Eluants were monitored atX=200–300 nm. Peptide samples were also analysed by DE MALDI-TOF massspectrometry (ThermoBioAnalysis Dynamo instrument). Anα-cyano-4-hydroxycinnamic acid matrix (Beavis, R. C. et al., (1992)Organic Mass Spectrometry 27, 156–158) was used and the appropriate m/zrange was calibrated using authentic peptide standards in the m/z range1,000–2,600.

Simultaneous Multiple Synthesis of Peptides

Peptides were synthesisted using a Multipin Peptide Synthesis Kit(Chiron Technologies Pty. Ltd., Clayton, VIC, Australia). Peptide chainswere assembled on “Macro Crowns” (SynPhase HM Series I, Rink AmideLinker; 5.3 μmol/crown) using Fmoc-amino acids (100 mM in DMF) andPyBOP/HOBt)/DIEA (1:1:1,5) coupling chemistry. The amino acid side-chainprotecting groups were 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Arg),trityl (Asn and Gln) and t-butyloxycarbonyl (Lys and Trp). Activatedamino acid solutions were dispensed using a PinAID device (ChironTechnologies). Coupling reactions were allowed to proceed for a minimumof 4 h. All other chain assembly manipulations, including repetitivedeprotection reactions (20% piperidine in DMF) and washing cycles (DMFand MeOH), were carried out according to procedures set out in the kitmanual. After coupling and deprotection of the N-terminal βAla residues,(+)-biotin (300 mM in DMF) was coupled (chemistry as above for aminoacids) during 4 h. After washing and drying, the “Macro Crowns” wereremoved from the synthesis device and placed into 10 mL cappedpolypropylene tubes. To each tube was added 1.5 mL ofcleavage/deprotection mixture. After 2 h, the “Macro Crowns” wereremoved and washed with 0.5 mL each of neat TFA. To each tube containingthe combined cleavage mixtures and washings Et₂O (8 mL) was added. Aftercooling to 4° C., the precipitated peptides were collected bycentrifugation (4 min at 5,000 r.p.m.) and decantation. The pellets wereresuspended in Et₂O (5 mL/tube). The suspensions were again cooled andthe peptides isolated as before. The washing process was repeated oncemore before the crude peptides were dried in vacuo.

The crude peptides were redissolved in 0.1% aq TFA using sonication (2mL/sample) and were applied to primed (MeOH then 0.1% aq TEA)solid-phase extraction cartridges (Merck LiChrolut RP-18, 500 mg). Thesewere successively washed (2×2 mL 0.1% aq TFA each) and eluted (2 mL 0.1%TFA in 6:4 MeCN/H₂O). The eluates were evaporated to dryness by vacuumcentrifugation.

Results and Discussion

The importance of eIF4E in translational regulation and cell growth isunderscored by observations which show that overexpression of eIF4Eleads both to increases in protein synthesis and to cellulartransformation in human and mouse cells (17,18). The mechanism by whicheIF4E overexpression leads to cell transformation is poorly understood.However, it is thought to be through the elevated translation of growthrelated mRNAs, which are normally translationally repressed (19). Inorder to study directly the role of eIF4E in cell transformation, aseries of experiments were carried out.

Human eIF4G₍₄₁₃₋₄₂₄₎ was conjugated to Penetratin, a known cell membranetranslocation peptide of sequence RQIKIWFQNRRMKWKK [SEQ ID NO: 6] (seepatent EP485578). Description of its synthesis and coupling to otherpeptides may be found in U.S. Pat. No. 5,888,762. The humaneIF4G₍₄₁₃₋₄₂₄₎-Penetratin conjugate was found to bind recombinant humaneIF4E in vitro (see FIG. 4). Surprisingly, wheat eIF4E₍₆₂₋₇₃₎ bound toand pulled down more recombinant human eIF4F in vitro than humaneIF4G₍₅₆₉₋₅₈₀₎ did (see FIG. 5). It was also observed that recombinanthuman 4E-BP1 competed with either human eIF4G₍₅₆₉₋₅₈₀₎ or wheateIF4G₍₆₂₋₇₃₎ for binding of recombinant human eIF4E in vitro (see FIG.6).

Wheat eIF4G₍₆₂₋₇₃₎ was found to inhibit cap-dependent translationinitiation, but not cap-independent translation initiation in vitro (seeFIG. 7). However, inhibition of cap dependent translation by eIF4Gpeptides was not detected in cultured mammalian cells. Furthermore, noinhibition of general translation by peptides from eIF4G or 4E-BP wasdetected in cultured mammalian cells.

Human eIF4G₍₅₆₉₋₅₈₀₎-Penetratin exhibited a cytotoxic or cytostaticeffect on selected cell lines (HaCaT cells, no effect observed withshort treatment (<24 h with 20 μM) but treatment of 60 h serum starvedcells began to die within 15 minutes of peptide treatment. Furthermore,human eIF4G₍₄₁₃₋₄₂₄₎-Penetratin and wheat eIF4G₍₆₂₋₇₃₎-Penetratin causedrapid cell death (possibly by apoptosis) in serum starved cell lines(see FIG. 8).

Resistance to Human eIF4G₍₅₆₉₋₅₈₀₎-Penetratin and wheateIF4G₍₆₂₋₇₃₎-Penetratin resulted from limited serum treatment (see FIGS.9 & 2). However, this serum induced resistance could be inhibited bypre-treatment with MEK inhibitor PD 098059 (see FIGS. 9 & 2).Furthermore, serum induced resistance could be mimicked by theoverexpression of a constitutively active MEK/ERK fusion (see FIGS. 10 &11). However, the serum induced resistance of cell lines was overcomeusing an increased concentration of peptide (72 h serum-starved MRC5cells died rapidly with addition of 10 μM 4G peptides; cells grown in10% serum show similar biological effect with 40 μM 4G peptides;however, control peptides (triple Ala substitution) were not cytotoxicat 40 μM).

Conservation of structure activity relationship (SAR) was found in wheatand human 4G peptides and human 4E-BP peptides in binding assay,functional cell free assay and cell culture assays.

In order to study directly the role of eIF4E in cell transformation, aseries of biotinylated synthetic peptides (Peptides synthesised byCyclacel) corresponding to the eIF4E interacting domain (binding motif)of human eIF4G, and wheat eIF4G and alanine substituted peptides thereofwere synthesised (see FIG. 12 a) and tested for their capacity tointeract with ³⁵S-Met labelled in vitro translated human eIF4E (NB.Peptides BP1 and BP1 YLM-AAA were not biotinylated). Peptides werecoupled to streptavidin coated agarose beads by a N-terminus linkedbiotin group and washed in PBS/0.2% Tween 3× before being incubated for1 hour at +4° C. with in vitro translated human eIF4E. Beads were washedas above and boiled for 5 min. in SDS loading buffer before the peptidebound proteins were separated on a 10% SDS gel. The bands werevisualised by autoradiography.

Triple alanine substituted derivatives such as HumaneIF4G_((569-580)Y416AL421AL422A) (see FIG. 8) were found not to inhibitcap-dependent translation initiation in vitro. However, 4G peptidescontaining specific single alanine substitutions (such as HumaneIF4G_((569-580)Y416A)) partially inhibited cap-dependent translationinitiation in vitro.

Triple alanine substituted derivatives such as HumaneIF4G_((569-580)Y416AL421AL422A)-Penetratin did not cause the observedbiological effect (apoptosis) in MRC5 cells (see FIG. 8). However, 4Gpeptides containing specific single alanine substitutions (such as HumaneIF4G_((569-580)Y416A)-Penetratin) had an intermediate biological effecton cultured mammalian cells, ie. reduced rate and extent of cell killingwas observed.

The three different wild type peptides were shown to interact with humaneIF4E and the H4G Y-A substitution had a lower binding affinity (seeFIG. 12 b(i)). Scrambled human eIF4G peptide, the triple alanine humaneIF4G peptide and the triple alanine wheat eIF4G peptides as well as thesingle H4G L-A did not interact with eIF4E. The Hu4G peptide also boundeIF4E in cell lysates whereas the Hu4G YLL-AAA variant did not (see FIG.12 b(ii)).

To investigate the effect of eIF4E binding peptides (eIF4G, BP1 and BP2)in living cells, 10 μM of the eIF4E binding peptide BP2 was incubatedwith serum-deprived or serum-fed MRC5 cells. Un-expectedly, the additionof the BP2 peptide to 72 h serum-starved MRC5 cells led to rapid celldeath (within 1 h) (FIG. 13 a, lane 4). In contrast, serum-fed cellswere insensitive to the effect of the BP2 peptide at this concentration(FIG. 13 a, lane 1). Incubation of either serum-fed or serum-starvedMRC5 cells with the triple alanine substitution peptide, BP2 YLL-AAA,had no significant effect on cell viability (FIG. 13 a, lane 5 and datanot shown). The sensitivity of the cells to the effect of the peptideincreased with the length of time the cells had been deprived of serum,with maximal effects observed by 72 h serum starvation (FIG. 13 a, lanes1–4). All subsequent experiments were therefore performed in cellsdeprived of serum for 72 h. Re-addition of 10% serum for one hour to 72h serum-starved cells protected them from the effects of the peptide(FIG. 13 a, lane 6). This protective effect could not be inhibited bypre-incubation of the cells with cycloheximide, an inhibitor of generalmRNA translation (FIG. 13 a, lane 7). This indicates that serum protectsagainst peptide-induced cell death through a post-translationalmodification rather than by inducing synthesis of new proteins, e.g.,cell survival proteins. To investigate further the effect ofeIF4E-binding peptides, serum-starved MRC5 cells were incubated withvarious concentrations of the BP2, BP1, Hu4G and W4G peptides (FIGS. 13b,c,d). Each of these peptides elicited rapid, dose-dependent, celldeath (within 30 min using 20 μM peptide and 1 h for 10 μM), whereas theaddition of the triple alanine substitution peptides, which are unableto bind eIF4E, had no significant effect on cell viability (FIGS. 13b,c,d). The singly alanine-substituted peptide, Hu4G Y-A, which hasreduced ability to bind eIF4E in vitro, also had a reduced capacity toinduce cell death (FIG. 13 c). Another single alanine substitutionpeptide, Hu4G L-A, which was unable to bind eIF4E in-vitro, had aseverely reduced ability to induce cell death up to 10 μM (FIG. 13 c).However, upon addition of higher concentrations (20 μM), this peptidecould induce cell death probably indicating some residual low bindingaffinity for eIF4E not detected in the pull-down assay (FIG. 13 c). Allthe peptides which are able to bind eIF4E in vitro can induce cell deatheven though they have very different sequences outside their commoneIF4E-binding motif (FIG. 12 a). In addition, peptides harbouring singleor triple alanine substitutions at conserved residues important inbinding to eIF4E either had no significant effect on cell survival or areduced ability to induce cell death. Taken together with the in vitrobinding studies, these data underpin a strong structure/activityrelationship and thus provide strong evidence that eIF4E-bindingpeptides induce cell death through their interaction with eIF4E.

During eIF4E-binding peptide-induced cell death, cells shrank andunderwent blebbing, two characteristics of apoptosis (FIG. 14 a). Toinvestigate whether eIF4E-binding peptide-induced cell death also causednuclear condensation and DNA cleavage, other characteristics associatedwith apoptosis, a number of methodologies were employed. FACS analysisof propidium iodide-stained MRC5 cells treated with the Hu4G peptiderevealed a shift in the DNA profile from G0/1 to sub G0/1, indicatingcell death and possible chromosomal DNA fragmentation/condensation (FIG.14 b). DNA fragmentation was confirmed using TdT-mediated dUTP nick endlabelling (TUNEL) (FIG. 14 c). Analysis of the cell nuclei by DAPIstaining revealed that the cells incubated with the Hu4G presented signsof nuclear condensation, either having condensed nuclei or nuclei with apunctate appearance (FIG. 14 d).

An early event considered decisive in apoptosis is the opening of themitochondrial permeability transition (MPT) pore (20–22). Tocharacterise further the eIF4E-binding peptide-induced cell death, thedevelopment of the MPT was investigated in living MRC5 cells loaded withthe fluorescent dye, JC1 (21). No changes in florescence were observedupon the addition of the inactive Hu4G YLL-AAA peptide (FIG. 14 e). Incontrast, addition of the Hu4G peptide led to a rapid increase (within 5min) in the intensity of the green fluorescence concomitantly with aloss of orange fluorescence indicative of a drop in Ψm within themitochondria due to the MPT pore opening (FIG. 14 e).

During apoptosis a conserved family of aspartic acid-specific cysteineproteases or caspases are frequently activated (23). However no suchactivation was detected in eIF4E-binding peptide-induced cell death(results not shown). Moreover, pre-treatment of the MRC5 cells withZVAD.fmk, a wide spectrum caspase inhibitor, did not affectpeptide-induced cell death (results not shown). Therefore, eIF4E-bindingpeptide-induced cell death appears not to involve caspase activation.Taken together, these data provide evidence that eIF4E peptide-inducedcell death in MRC5 cells proceeds through a caspase-independentmechanism which exhibits a number of features observed in apoptosis. Therapidity with which the cells die and apparent lack of caspaseactivation are not features associated with classical apoptosis.However, it is clear that the activation of caspases is not aprerequisite for apoptosis (24,25). For example, it has been reportedthat mitochondrial associated protein, apoptosis inducing factor (AIF),can induce rapid caspase-independent apoptosis (26). The effect of theseeIF4E-binding peptides on cell survival was also tested on a number ofother cell-types including HaCaT, Swiss 3T3, RATI and IIeLa cells. Inall of cases, addition of the Hu4G peptide resulted in rapid cell deathwhereas the Hu4G YLL-AAA peptide had no effect on cell survival (resultsnot shown). However, characterisation of the cell death process in thesecells was not investigated in detail.

In the presence of 10% ECS, cells were resistant to treatment with 20 μmof the peptides. Cells only died if they were serum deprived for 72hours (more than 85%) within 15 minutes after the peptide had beenapplied (see FIG. 15). However, if serum deprived cells (72 h) werepre-treated with 10% FCS or with 20 nM PMA (phorbol ester) for 15minutes before the peptides were added, the cells survived thesubsequent peptide treatment (60–70%).

Furthermore, if the serum deprived cells were instead pre-treated withthe MAPK inhibitor PD098059 for 1 hour before 10% FCS was added,approximately 80–90% of the cells died. This result shows that celldeath is linked to a genetic program and that the cells can be rescuedfrom peptide induced death by addition of FCS or PMA. It is alsosuggested by the speed with which the cells died after peptide treatmentand the rapid rescue by FCS or PMA and the effect of the MAPK inhibitor,that the effect of the peptides on cell death is dependent on secondarymodifications in the cells.

Serum deprived cells were treated with the general translationinhibitors Cyclohexamide or Pactamycin at indicated concentrations orthe H4G peptide in the presence of 35S-Met for 30 minutes (see FIGS. 16a,b). Cells were lysed and the amount of translation was estimated bycounting incorporated 35S-Met in precipitated protein fractions. Asexpected, the peptide treated cells do not incorporate 35S-Met and theydie. However, general translation inhibitors block translation but theydo not kill the cells.

It remained possible that eIF4E-binding peptide-induced cell deathinvolved the up- or down-regulation of the translation of a specificmRNA or subset of mRNAs. To investigate this, MRC5 cells were treatedwith cycloheximide or pactamycin to prevent ongoing protein synthesisprior to the addition of the Hu4G peptide. However, this did not resultin any protection against the effect of the Hu4G peptide (FIG. 16 c).These data show that continued translation is not required for thepeptides to induce cell death, and thus provides evidence that the up-or down-regulation of the translation of a specific mRNA(s) does notmediate eIF4E-binding peptide-induced cell death.

In another experiment, cells that were serum deprived for 72 hours andpre-treated with general translation inhibitors were shown to be just assusceptible to cell death (85%) as cells not treated with translationinhibitors. This strongly indicates that the effect of cell killing bythe peptides is not mediated by inhibition of translation and istherefore not mediated by a translation product. This observation isvery surprising and novel.

The present data thus indicate that eIF4E plays a direct role incontrolling cell survival that is not linked to its known role inregulating mRNA translation. It is presently not clear what mechanismunderlies this eIF4E-binding peptide-induced cell death. Without wishingto be bound by theory it is possible that it is associated with a yetundefined function of eIF4E. Recently, it has been reported that eIF4Eco-localises in the nucleus with splicing factors and eIF4E maytherefore play an additional role in splicing or RNA export (27). Aspenetratin-linked peptides can enter all compartments of the cell it ispossible that these peptides interfere with a nuclear function of eIF4Ewhich results in cell death. However, it is also possible thatdeleterious perturbations in eIF4E function may directly trigger theapoptotic machinery. This could be a “checkpoint” mechanism by which thecells sense the integrity of the translation machinery. Indeed, therapidity of cell death suggests that binding of the peptides to eIF4Emay directly signal the induction of cell death.

In conclusion, the present data clearly indicates that eIF4E plays acritical role in cell survival, which may be related to its known rolein cell transformation. However, its role in cell survival appears toinvolve a novel mechanism independent of its known function in mRNAtranslation.

REFERENCES

-   1. Sonenberg, N. & Gingras, A. The mRNA 5′ cap-binding protein eIF4E    and control of cell growth. Curr Opin Cell Biol 10, 268–75 (1998).-   2. Hentze, M. EIF4G: a multipurpose ribosome adapter? Science 275,    500–1 (1997).-   3. Pyronnet, S. et al. Human eukaryotic translation initiation    factor 4G(eIF4G) recruits mnkl to phosphorylate eIF4E. EMBO J. 18,    270–9 (1999).-   4. Lawrence Jr, J. & Abraham, R., R. PHAS/4E-BPs as regulators of    mRNA translation and cell proliferation. Trends Biochem Sci 22,    345–9 (1997).-   5. Rousseau, D., Gingras, A., Pause, A. & Sonenberg, N. The    eIF4E-binding proteins 1 and 2 are negative regulators of cell    growth. Oncogene 13, 2415–20 (1996).-   6. Flynn, A. & Proud, G. Insulin-stimulated phosphorylation of    initiation factor 4E is mediated by the MAP kinase pathway. FEBS    Lett 389, 162–6 (1996).-   7. Rosenwald, I., Laxaris-Karatzas, A., Sonnenberg, N. &    Schxnidt, E. Elevated levels of cyclin D1 protein in response to    increased expression of eukaryotic initiation factor 4E. Mol Cell    Biol 13, 7358–63 (1993).-   8. Shantz, L., Hu. R. & Pegg, A. Regulation of ornithine    decarboxylase in a transformed cell line that overexpresses    translation initiation factor eIF-4E. Cancer Res 56, 3265–9 (1996).-   9. Li, B., Liu, L., Dawson, M. & De Benedetti, A. Overexpression of    eukaryotic initiation factor 4E(eIF4E) in breast carcinoma. Cancer    79, 2385–90 (1997).-   10. Rosenwald, J. B. et al. Upregulation of protein synthesis    initiation factor eIF4E is an early event during colon    carcinogenesis. Oncogene 18, 2507–2517 (1999).-   11. Li, B., McDonald, J., Nassar, R. & De Beneditte, A. Clinical    outcome in stage I to III breast carcinoma and eIF4E overexpression.    Ann Surg 227, 756–61; discussion 761–3 (1998).-   12. Nathan, C. et al. Detection of the proto-oncogene eIF4E in    surgical margins may predict recurrence in head and neck cancer.    Oncogene 15, 579–84 (1997).-   13. De Benedetti, A. & Rhoads, R. Overexpression of eukaryotic    protein synthesis initiation factor 4E in HeLa cells results in    aberrant growth and morphology. Proc Natl Acad Sci USA 87, 8212–6    (1990).-   14. Fukuchi-Shimogori, T. et al. Malignant transformation by    overproduction of translation initiation factor eIF4G. Cancer Res    57, 5041–4 (1997).-   15. Polunovsky, V. et al Translation control of programmed cell    death: eukaryotic translation initiation factor 4E blocks apoptosis    in growth-factor-restricted fibroblasts with physiologically    expressed or deregulated Myc. Mol Cell Biol 16, 6573–81 (1996).-   16. Fletcher, C. et al, 4E binding proteins inhibit the translation    factor without folded structure. Biochemistry 37, 9–15 (1998).-   17. Lawrence Jr, J. & Abraham, R. PHAS/4E-BPs as regulators of mRNA    translation and cell proliferation. Trends Biochem Sci 22, 345–9    (1997).-   18. Rousseau, D., Gingras, A., Pause, A. & Sonenberg, N. The    eIF4E-binding proteins 1 and 2 are negative regulators of cell    growth. Oncogene 13, 2415–20 (1996).-   19. Sonenberg, N. & Gingras, A. The mRNA 5′ cap-binding protein    eTF4E and control of cell growth. Curr Opin Cell Biol 10, 268–75    (1998).-   20. Green, D. R., Reed, J. C. Mitochondria and apoptosis.    Science 1998. 281; 1309–1312.-   21. Kroemer, G: The proto-oncogene Bcl-2 and its role in regulating    apoptosis. Nat Med 1997. 3; 614–620.-   22. Minamikawa, T., Williams, D. A., Bowser, D. N., Nagley, P:    Mitochondrial permeability transition and swelling can occur    reversibly without inducing cell death in intact human cells. Exp    Cell Res 1999. 246; 26–37.-   23. Wolf, B. B., Green, D. R: Suicidal tendencies: apoptotic cell    death by caspase family proteinases. J Biol Chem 1999. 274;    20049–20052.-   24. Okuno, S., Shimizu, S., Ito, T., Nomura, M., Hamada, E.,    Tsujimoto, Y., Matsuda, H: Bcl-2 prevents caspase-independent cell    death. J Biol Chem 1998. 273; 34272–34277.-   25. Xiang, J., Chao, D. T., Korsmeyer, S. J: BAX-induced cell death    may not require interleukin 1 beta-converting enzyme-like proteases.    Proc Natl Acad Sci USA 1996. 93; 14559–14563.-   26. Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B.    E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P.,    Loeffler, M., Larochette, N., Goodlett, D. R., Aebersold, R.,    Siderovski, D. P., Penninger, J. M., Kroemer, G: Molecular    characterization of mitochondrial apoptosis-inducing factor.    Nature 1999. 397; 441–446.-   27. Dostie, J., Lejbkowicz, F., Sonenberg, N: Nuclear Eukaryotic    Initiation Factor 4E (eIF4E) Colocalizes with Splicing Factors in    Speckles. J Cell Biol 2000. 148; 239–246.

1. A therapeutic method of inducing programmed cell death, said method comprising administering to a recipient a peptide of 10–25 amino acids, comprising the sequence: (KR)xxYxxx(F/Q)L(L/M) (SEO ID NO:5), wherein x is any amino acid.
 2. The method according to claim 1, wherein said peptide comprises the sequence: KKRYDREFLLGF (SEQ ID NO:1); RVRYSRDOLLDL (SEQ ID NO:2); or RIIYDRKFL(L/M) (SEQ ID NO:3).
 3. The method according to claim 1, wherein said method induces cell death in tumour cells.
 4. A method of inducing programmed cell death, said method comprising administering to a recipient a peptide of 10–25 amino acids in length, comprising the sequence: (K/R) xxYxxx (F/Q) L (L/M) (SEQ ID NO:5), wherein x is any amino acid, a synthetic amino acid or an unnatural amino acid.
 5. A method of inducing programmed cell death in tumour cells, said method comprising administering to a recipient a peptide of 10–25 amino acids in length, comprising the sequence: (K/R) xxYxxx (F/Q) L (L/M) (SEQ ID NO:5), wherein x is any amino acid, a synthetic amino acid or an unnatural amino acid. 